CN106170493A - The novel purification process of promoting sexual gland hormone - Google Patents
The novel purification process of promoting sexual gland hormone Download PDFInfo
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- CN106170493A CN106170493A CN201580019503.6A CN201580019503A CN106170493A CN 106170493 A CN106170493 A CN 106170493A CN 201580019503 A CN201580019503 A CN 201580019503A CN 106170493 A CN106170493 A CN 106170493A
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- sexual gland
- purification
- promoting sexual
- gland hormone
- chromatography
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- 238000000746 purification Methods 0.000 title claims abstract description 70
- 229940088597 hormone Drugs 0.000 title claims abstract description 44
- 239000005556 hormone Substances 0.000 title claims abstract description 44
- 210000004907 gland Anatomy 0.000 title claims abstract description 43
- 230000001737 promoting effect Effects 0.000 title claims abstract description 42
- 230000001568 sexual effect Effects 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 37
- 238000004440 column chromatography Methods 0.000 claims abstract description 31
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 25
- 239000000203 mixture Substances 0.000 claims abstract description 22
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 19
- 230000003993 interaction Effects 0.000 claims abstract description 19
- 238000011210 chromatographic step Methods 0.000 claims abstract description 8
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 claims description 34
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 claims description 34
- 229940028334 follicle stimulating hormone Drugs 0.000 claims description 34
- 238000011026 diafiltration Methods 0.000 claims description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 238000005349 anion exchange Methods 0.000 claims description 16
- 239000000758 substrate Substances 0.000 claims description 14
- 239000000872 buffer Substances 0.000 claims description 12
- 238000005571 anion exchange chromatography Methods 0.000 claims description 11
- 235000002639 sodium chloride Nutrition 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 9
- 239000011543 agarose gel Substances 0.000 claims description 8
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- 238000001728 nano-filtration Methods 0.000 claims description 6
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- 102000011022 Chorionic Gonadotropin Human genes 0.000 claims description 5
- 108010062540 Chorionic Gonadotropin Proteins 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 229940084986 human chorionic gonadotropin Drugs 0.000 claims description 5
- 238000001471 micro-filtration Methods 0.000 claims description 5
- 230000007935 neutral effect Effects 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- 238000004113 cell culture Methods 0.000 claims description 3
- 238000004255 ion exchange chromatography Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
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- 239000008351 acetate buffer Substances 0.000 claims description 2
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical group N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
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- 235000011130 ammonium sulphate Nutrition 0.000 claims description 2
- 239000007979 citrate buffer Substances 0.000 claims description 2
- 239000008362 succinate buffer Substances 0.000 claims description 2
- 235000019270 ammonium chloride Nutrition 0.000 claims 1
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- 102000004169 proteins and genes Human genes 0.000 abstract description 14
- 230000006872 improvement Effects 0.000 abstract description 2
- 238000005342 ion exchange Methods 0.000 abstract description 2
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- 239000000243 solution Substances 0.000 description 13
- 239000000975 dye Substances 0.000 description 10
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- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000008215 water for injection Substances 0.000 description 2
- CVOFKRWYWCSDMA-UHFFFAOYSA-N 2-chloro-n-(2,6-diethylphenyl)-n-(methoxymethyl)acetamide;2,6-dinitro-n,n-dipropyl-4-(trifluoromethyl)aniline Chemical compound CCC1=CC=CC(CC)=C1N(COC)C(=O)CCl.CCCN(CCC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O CVOFKRWYWCSDMA-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 101100230376 Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) celI gene Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000006771 Gonadotropins Human genes 0.000 description 1
- 108010086677 Gonadotropins Proteins 0.000 description 1
- 206010058359 Hypogonadism Diseases 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 0.000 description 1
- 206010033266 Ovarian Hyperstimulation Syndrome Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
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- 238000012239 gene modification Methods 0.000 description 1
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- 235000013617 genetically modified food Nutrition 0.000 description 1
- 239000002622 gonadotropin Substances 0.000 description 1
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- 201000003368 hypogonadotropic hypogonadism Diseases 0.000 description 1
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- 229910052751 metal Inorganic materials 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/32—Bonded phase chromatography
- B01D15/325—Reversed phase
- B01D15/327—Reversed phase with hydrophobic interaction
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
- B01D15/361—Ion-exchange
- B01D15/363—Anion-exchange
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Endocrinology (AREA)
- Reproductive Health (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Water Supply & Treatment (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
Present invention provide for the method for the improvement of the desired promoting sexual gland hormone of purification from the crude mixture comprising at least one contaminating protein.The method of the desired promoting sexual gland hormone of purification according to the present invention includes, before using any column chromatography steps to be used for being further purified, uses the affinity chromatography as the first column purification step.This purification process may further include ion exchange and/or hydrophobic interaction chromatographic step to obtain the promoting sexual gland hormone albumen with expectation subtype distribution of substantially purification.
Description
Technical field
Present invention provide for purification desired rush gonad from the crude mixture comprising at least one contaminating protein to swash
The method of the improvement of element.The method of the desired promoting sexual gland hormone of purification according to the present invention includes, is using any column chromatography step
Suddenly, before being used for being further purified, affinity chromatography (affinity chromatograph, affinity as the first column purification step are used
chromatography).This purification process may further include ion exchange and/or hydrophobic interaction chromatographic step
To obtain the promoting sexual gland hormone albumen with expectation subtype distribution of substantially purification.
Background technology
Follicle stimulating hormone is comprise α subunit (subunit) (92 aminoacid) and β subunit (111 aminoacid) heterogeneous
Dimeric glycoprotein.Glycosylation occurs on the specific position of α and β subunit.Follicle stimulating hormone controls in women
The growth of ovary follicle, and show important effect in the spermatogenesis of induction man.Follicle stimulating hormone be expressed as with
Lower therapeutic use-
-woman is not ovulated
The ovarian hyperstimulation of-control is induced for the multiple filter in the women of external fertilization (IVF)/embryo transfer (ET)
The growth of bubble
-follicle stimulating hormone is combined with LH the follicular development recommended to be used for stimulate in woman
-in male, utilize adjoint hCG therapy for hypogonadotropic hypogonadism.
The present inventor by r-DNA technology use genetic modification Chinese hamster ovary celI as host system, the most intrinsic
Develop recombinant r-hFSH or follitropin.
The present invention relates to the purification of promoting sexual gland hormone.There are some in the prior art for purification promoting sexual gland hormone
The purification process known.Such purification process includes using high-efficient liquid that is expensive and that need a large amount of organic solvent during operation
Phase chromatograph (HPLC) (such as, patent documentation WO2006/051070).By HPLC purification at industrial scale one or more
In the case of promoting sexual gland hormone, much more excessively the high cost of instrument and requirement organic solvent are main restrictions.
WO2007/065918 discloses for purification FSH or the method for FSH mutant, including make to comprise described FSH or
The liquid of FSH mutant stands below step: (1) dye affinity chromatography；(2) Weak anion-exchange chromatography (3) hydrophobicity phase
Interaction chromatograph；And (4) strong anion exchange chromatographic；It can be carried out in any order.The affine color of dyestuff in the first step
Before spectrum purification step, it includes the optional step of the capture step (capture step) as step (0).
Dye affinity chromatography is for the egg of the affinity of the binding site on numerous protein based on fixed dye
White purification step.This chromatographic technique is nonspecific.Fixed dye can non-specifically be bound to glycosylation albumen
Molecule.The further drawback of this purification technique is, may have the albumen of other similar types being present in crude mixture and grind
The chance of the co-elute together of the albumen in studying carefully.Additionally, there is also the possibility of dye molecule or its part and expectation part co-elute
Property.So, it does not provide the level of the gratifying purity expecting albumen.The major defect of these synthetic dyestuffs is, right
Selection course in specific biomolecule is experiential, and needs to screen on a large scale during method is developed
Journey.Meanwhile, the present invention does not include dye affinity chromatography step.Therefore, in purification process described herein, it is to avoid dyestuff or
The chemical contamination of modified dye.
WO 2005/063811 discloses for purified recombinant mankind FSH or the method for FSH variant, exchanges including ion
Chromatograph；Immobilized metal chromatograph；And the step of hydrophobic interaction chromatograph (HIC), it can enter in any order
OK.
The method for purification promoting sexual gland hormone described in the present invention does not include using HPLC.Therefore, the present invention
Disclose simple, cost-effective, height can scale, industry is feasible and the purification process of environmental benefits is to obtain height
The promoting sexual gland hormone of degree purification.The disclosedest purification process can be used for purification rush gonad from crude mixture and swashs
The mixture of element.
It is an object of the present invention to provide new, advantageous approach for purified recombinant FSH or its functional variant thereof.?
In the present invention, have been disclosed for the novel method for purified recombinant mankind's follicle stimulating hormone, the most do not use HPLC method
Step.
Summary of the invention
Present invention provide for the method for purification promoting sexual gland hormone from crude mixture.Crude mixture can comprise except
The albumen of the pollution outside desired albumen, interior immunogenic peptide, product related species and other impurity.
In an aspect, the invention provides the method for purification promoting sexual gland hormone from crude mixture, it includes that one is
The chromatographic step not including HPLC of row.
In one embodiment, by the present invention in that with affinity column chromatography, first capture, then from post eluting, there is height
The albumen of purity level, it is provided that the method being derived from the promoting sexual gland hormone of cell culture from crude mixture purification.Crude mixture
Can include being derived from the relevant material of the contaminating protein of host cell, product and other impurity in addition to the albumen in research.
The present invention have also demonstrated and removes most host cell contaminating protein by affinity chromatography, has maximum simultaneously
Under the neutral buffered liquid pH condition of the response rate or condition of acidic pH, the albumen in eluting research leaves post.
In one embodiment, the present invention have also demonstrated after affinity column eluting, under neutrality or condition of acidic pH
Keep at least about 24 hours constant, such as the HP-SEC assessment by analyzing, it is desirable to the molecule of promoting sexual gland hormone albumen complete
Property.
In one embodiment, present invention also offers and with binding pattern, there is the phase by Anion exchange column chromatography
Hope the purification of the promoting sexual gland hormone of hypotype.
In another embodiment, the invention provides with combination-elution mode use hydrophobic interaction post color
Compose and from desired protein fraction, remove the impurity relevant with product that the technique of residual is correlated with.In relatively low electrical conductivity
(conductance) under with line form or in a step-wise fashion, carry out expecting the eluting of albumen.
In one preferred embodiment, the expectation promoting sexual gland hormone of crude mixture is carried out being derived from according to following steps
Purification:
1. affinity chromatography
2. Anion exchange column chromatography, be followed by being available from those skilled in the art knowledge and do not include HPLC's
Other suitable purification techniques.
In another embodiment, carry out being derived from the expectation promoting sexual gland hormone of cell culture according to following purification step
Purification:
1. affinity chromatography
2. Anion exchange column chromatography
3. hydrophobic interaction chromatograph
After affinity chromatography step, hydrophobic interaction chromatographic step can be carried out in any order.By obtaining
Derive from the knowledge of those skilled in the art and do not include any purification technique of HPLC, can carry out in this application further
The method of described purification.
This purification technique includes diafiltration, any column chromatography, nanofiltration or any other known purification technique.
The abbreviation used in this manual is defined as follows:
Affinity substrate: affinity column purification
AEX: Anion exchange column chromatography
DF: diafiltration
HIC: hydrophobic interaction column chromatography
HP-SEC: high-performance-size exclusion chromatography
HPL: high performance liquid chromatography
U-HCG: urine HCG
U-FSH: urine FSH
MWCO: weight shutoff
NaCl: sodium chloride
UF: ultrafiltration
WFI: water for injection
Accompanying drawing explanation
Fig. 1 shows by the affinity column chromatography step used in purge process from the washing of r-hFSH of crude mixture
De-curve.
Fig. 2 shows the polypeptide distribution of the r-hFSH of the affinity column eluting by non-reduced SDS-PAGE.
Fig. 3 shows the elution curve of the r-hFSH by the AEX column chromatography steps used in purge process.
Fig. 4 shows the purity of the r-hFSH of the anion exchange column purification by HP-SEC.The figure shows r-hFSH
Simple spike purity.
Fig. 5 shows the elution chromatography curve of the r-hFSH by the HIC chromatographic step used in purge process.
Fig. 6 shows the purity of the r-hFSH of the HIC purification by the HP-SEC analyzed.The figure shows the list of r-hFSH
One peak purity.
Fig. 7 shows the purity of the r-hFSH drug substance by HP-SEC.
Fig. 8 shows by the affinity column chromatography step used in purge process from the washing of u-HCG of crude mixture
De-curve.
Fig. 9 shows the elution curve of the u-HCG by the AEX column chromatography steps used in purge process.
Figure 10 shows the polypeptide distribution of the u-HCG by non-reduced SDS-PAGE.
Figure 11 shows the polypeptide distribution of the SDS-PAGE of the u-FSH by purification.
Figure 12 shows the purity of the u-FSH by HP-SEC.
Detailed description of the invention
Present invention provide for the novel purification process of the preferred FSH of desired promoting sexual gland hormone or its functional variant thereof.
In one embodiment, the invention provides the purification of one or more promoting sexual gland hormone from crude mixture
Method, including using the first affinity chromatography, uses other column chromatography steps not including HPLC subsequently.Crude mixture can comprise
Contaminating protein, interior immunogenic peptide, product related species and other impurity in addition to expectation albumen.
In one embodiment, present invention provide for the novel method of one or more promoting sexual gland hormone of purification,
Including using affine and ion exchange chromatography step.Ion exchange chromatography can be Anion exchange column chromatography or cation friendship
Change column chromatography.
In one embodiment, for the base for post matter of affinity chromatography step selected from FSH is specific and promoting sexual gland hormone
Specificity affinity substrate (gonadotropin-specific affinity matrix).In another embodiment, it is used for
The base for post matter of anion-exchange chromatography step is selected from DEAE agarose gel, Mono Q and Q agarose gel XL, preferably Q agar
Sugar gel.
In one preferred embodiment, the purification process of one or more promoting sexual gland hormone includes following chromatograph step
Rapid:
1. affinity chromatography
2. anion exchange or cation exchange column chromatography
3.HIC chromatograph
This column chromatography steps can be carried out in any order.
In another embodiment, the invention provides with combination-elution mode use hydrophobic interaction post color
Compose and from desired protein fraction, remove the impurity relevant with product that the technique of residual is correlated with.Utilize dense to the salt of Gradient
Degree (down-the-gradient salt concentration) carries out the eluting of the desired albumen with main peak form.
In further embodiment, the base for post matter for hydrophobic interaction chromatograph is selected from phenyl Sepharose
(phenyl sepharose), Butyl Sepharose, Octyl Sepharose, preferably phenyl Sepharose.
In embodiment in addition, for the salt choosing of the desired albumen of eluting in hydrophobic interaction chromatographic step
From ammonium sulfate, sodium chloride, ammonia chloride and sodium sulfate, preferably sulfuric acid ammonium.
In an embodiment more preferably, carry out one or more rush from crude mixture according to following steps
The purification of sex gland hormones:
-step 1: cell separation and readjusting
-step 2: affinity column chromatography
-step 3: virally inactivated
-step 4: Ultrafiltration-Diafiltration and readjust (UF/DF)
-step 5: Anion exchange column chromatography (AEX)
-step 6: readjust
-step 7: hydrophobic interaction column chromatography (HIC)
-step 8: Ultrafiltration-Diafiltration
-step 9: remove virus by nanofiltration
-step 10: microfiltration (microfiltration)
-step 11: under freezing conditions store
In another embodiment, in the case of not using HIC chromatographic step, can carry out being derived from crude mixture
The purification of desired promoting sexual gland hormone.
In further embodiment, filtration media is selected from water, Tris-Cl buffer, citrate buffer, phosphoric acid
Salt buffer, Succinate Buffer, acetate buffer and a combination thereof.
In one preferred embodiment, promoting sexual gland hormone is selected from follicle stimulating hormone (FSH), lutropin
(LH), human chorionic gonadotropin (HCG) and its suitably combine.
In an embodiment more preferably, promoting sexual gland hormone is selected from r-hFSH, u-FSH, r-hLH, u-LH, r-
HHCG and u-HCG.
Column chromatography steps according to the present invention is describing in further detail below:
I) affinity column chromatography:
After readjusting, the supernatant of clarification is made to pass through promoting sexual gland hormone specific affinity column substrate to capture choosing
Selecting property from the desired promoting sexual gland hormone of crude mixture.Before the desired albumen of eluting, affinity substrate stands intermediate column
Washing.By the desired albumen of post eluting under about neutral pH.
II) Anion exchange column chromatography
After diafiltration, will be used on the solution loadings that recombinant follicle stimulating hormone be comprised to anion-exchange column further
Ground purification has the desired albumen of expectation subtype distribution.Carry out this post step with combination-elution mode, and carry out this post step
Suddenly it is mainly used in removing the less desirable hypotype of recombinant follicle stimulating hormone, concurrently separates the described albumen with expectation hypotype.
By in protein load to the post at about pH 8.0 to be bound to substrate.With identical level pad (equilibration
Buffer) column scrubber substrate is to remove unconjugated pollutant.After equilibration buffer solution, buffer with less than initial balance
The buffer of the pH of liquid pH carries out second time and washs.Subsequently, in the presence of NaCl, carry out third time at acidic to wash.With
Level pad reequilibrate post, and the eluting of desired albumen is carried out with the electrical conductivity increased.In order to implement according to the present invention
Anion-exchange chromatography, it is also possible to other anionites used can be selected from DEAE agarose gel, Mono Q,
Q agarose gel XL etc..Anionite Q agarose gel has been used to the present invention.
III) hydrophobic interaction column chromatography:
With combination-elution mode, by hydrophobic interaction column chromatography, carry out from containing at least one less desirable dirt
Purification desired promoting sexual gland hormone albumen in the mixture of dye thing.After completing albumen-be loaded on post, utilize downwards-ladder
The salinity of degree i.e. has the electric conductivity of reduction compared with level pad electric conductivity, the desired promoting sexual gland hormone of eluting from post
Albumen.The eluting of desired promoting sexual gland hormone albumen occurs with the form of simple spike.With the albumen of fraction collection eluting and contain
The fraction having desired purity level is aggregated together.For carrying out hydrophobic interaction column chromatography according to the present invention, can
To use HIC resin, such as phenyl Sepharose, Butyl Sepharose 4FF, Octyl Sepharose etc..
Analytical technology for the present invention:
HP-SEC: by using the TSK-3000 post of the sodium phosphate buffer balance utilizing the pH 6.7 comprising sodium sulfate,
The size exclusion chromatography (HP-SEC) being analyzed.Isocratic-pattern eluted protein with 0.5mL/min.
Purification step according to the present invention is elaborated further below:
Embodiment:
Here, utilize following non-limiting examples to illustrate the present invention, these non-limiting embodiments should not be solved
It is interpreted as limiting by any way the scope of the present invention:
Embodiment 1: the purification of recombinant FSH
Step 1: cell separation and readjusting
After results in batches, in order to obtain containing the albumen in studying together with the clarified supernatant of other solvable pollutant
Liquid, first passes through centrifugation and then depth-type filtration, separates cell from culture meat soup.In about 10,000g × 30 minute
Lower enforcement centrifugation.Pass throughThe film of 0.45 0.22 μm carries out depth-type filtration for the most fining.By clarification
Supernatant readjusts has next affinity column level pad condition, such as pH and electrical conductivity to adjust.When by purification from urine
During the promoting sexual gland hormone obtained, it is not required that the step for.
Step 2: affinity column chromatography
After readjusting, the supernatant of clarification is made to capture desired albumen by affinity column substrate with selectivity.?
Before the desired albumen of eluting, affinity substrate stands intermediate column washing.By the desired albumen of post eluting in about neutral pH.Post color
Spectral curve shows in FIG.When being analyzed by SDS-PAGE as shown in Figure 2, the albumen of affinity purification shows
Single band purity in gel.
Step 3: Ultrafiltration-Diafiltration and readjusting
Use the 10kDa MWCO membrane filter of the Tris-Cl buffer of the pH 7.0 for low ionic strength, pass through
UF/DF readjusts the albumen of affinity column eluting, matching with next post (Q post) step level pad condition (such as, pH and
Electrical conductivity).Before loading on Q post, make the protein solution filter by 0.22 μm of diafiltration.
Step 4: virally inactivated
By the protein solution incubation about 4-of diafiltration under the conditions of identical pH in the presence of solvent/detergent or detergent
6 hours, the most continuously stirred for virally inactivated.
Step 5: Anion exchange column chromatography (AEX)
After diafiltration, will be used on the solution loadings that recombinant follicle stimulating hormone be comprised to anion-exchange column further
Ground purification has the desired albumen of expectation subtype distribution.Carry out this post step with combination-elution mode, and carry out this post step
Suddenly it is mainly used in removing the less desirable hypotype of recombinant follicle stimulating hormone, concurrently separates the described albumen with expectation hypotype.
Column chromatography curve shows in figure 3.
By in protein load to the post at about pH 8.0 to be bound to substrate.By identical equilibration buffer solution base for post matter
To remove unconjugated pollutant.After equilibration buffer solution, with the buffer of the pH less than initial balance pH of buffer
Carry out second time to wash.Subsequently, in the presence of NaCl, carry out third time at acidic to wash.Use level pad reequilibrate
Post, and the eluting of desired albumen is carried out with the electrical conductivity increased.
Such as being evaluated by HP-SEC of figure 4 illustrates, after Q post step, it was observed that desired recombinant FSH egg
White purity is more than 98%.
Step 6: readjust
Before loading on HIC post, the protein solution of diafiltration is mixed with the sodium chloride solution of concentration to adjust further
Whole have HIC column equilibration condition and by the membrane filter of 0.22 μm.
Step 7: hydrophobic interaction column chromatography (HIC)
After readjusting, make the protein solution comprising expectation albumen by hydrophobic interaction chromatography matrix, use
In being further purified with combination-elution mode.After being bound to base for post matter, under relatively low electrical conductivity with line form or
In a step-wise fashion, eluted protein.Column chromatography curve shows in Figure 5.Collect the main eluting comprising recombinant follicle stimulating hormone
Peak is used for being processed further.As shown in fig. 6 evaluated by HP-SEC, after the 3rd post step, it is thus achieved that be more than
The desired recombinant FSH of 99% purity.
Step 8: Ultrafiltration-Diafiltration
After the 3rd post step, at ambient temperature, the solution comprising recombinant follicle stimulating hormone stands Ultrafiltration-Diafiltration
Step is used for buffer-exchanged.
Step 9: nanofiltration
After buffer exchange step, recombinant follicle stimulating hormone stands nanofiltration step, for virus sweep.As
Evaluated by HP-SEC, during and after nanofiltration step, do not observe significant loss of proteins or accumulation.In nanometer
After filtration, it was observed that the purity of recombinant follicle stimulating hormone is more than 99%.
Step 10: microfiltration
Finally, make the recombinant follicle stimulating hormone solution of the purification aseptic membrane filter by 0.22 μm, and with
Concentration between 0.2mg/mL and 2.5mg/mL stores (for short-term storage) or at freezing conditions under cryogenic with liquid
Down for longer-term storage.
Such as being evaluated by HP-SEC of figure 7 illustrates, final the most after purification, it was observed that the purity of recombinant FSH is extremely
It is 99% less.
After final purification, it was observed that the subtype distribution of the recombinant FSH albumen of purification is similar to standard.
Embodiment 2: the purification of urine HCG
Step 1: affinity column chromatography
After readjusting, u-HCG crude mixture is made to capture desired albumen by affinity column substrate with selectivity, and
And hereafter eluting.Before the desired albumen of eluting, affinity substrate stands intermediate column washing.By the post eluting phase at acidic
The albumen hoped.Column chromatography curve shows in fig. 8.
Step 2: Ultrafiltration-Diafiltration and readjusting
Use the 10kDa MWCO membrane filter of the buffer of the pH 7.0 for low ionic strength, by UF/DF weight
The new albumen regulating affinity column eluting, to match (such as, pH and conductance with next post (Q post) step level pad condition
Rate).Before loading on Q post, make the protein solution filter by 0.22 μm of diafiltration.
Step 3: virally inactivated
By the protein solution incubation about 4-of diafiltration under the conditions of identical pH in the presence of solvent/detergent or detergent
6 hours, the most continuously stirred for virally inactivated.
Step 4: Anion exchange column chromatography (AEX)
After diafiltration, have being used for purification further on the solution loadings comprising u-HCG to anion-exchange column
The desired albumen of desired subtype distribution.Carry out this post step with combination-elution mode, and carry out this post step and mainly use
In the less desirable hypotype of removal recombinant follicle stimulating hormone, concurrently separate the described albumen with expectation hypotype.Column chromatography is bent
Line shows in fig .9.By in protein load to the post at about pH 8.0 to be bound to substrate.With identical equilibration buffer solution
Base for post matter is to remove unconjugated pollutant.After equilibration buffer solution, with the pH's less than initial balance pH of buffer
Buffer carries out second time and washs.Subsequently, in the presence of NaCl, carry out third time at acidic to wash.Use level pad
Reequilibrate post, and the eluting of desired albumen is carried out with the electrical conductivity increased.
As figure 10 illustrates, after Q post step, observe single band purity by SDS PAGE.
Step 5: Ultrafiltration-Diafiltration
After the 3rd post step, at ambient temperature, the solution comprising u-HCG stands Ultrafiltration-Diafiltration step for delaying
Rush liquid exchange.
Step 7: microfiltration
Finally, make the u-HCG solution of the purification aseptic membrane filter by 0.22 μm, and with 0.2mg/mL and
Concentration between 2.5mg/mL stores (for short-term storage) or under freezing conditions for long-term under cryogenic with liquid
Storage.
After final purification, it was observed that the subtype distribution of the u-HCG of purification is similar to standard u-HCG.
Embodiment 3: the purification of urine FSH
The purification process of u-FSH is carried out in the way of describing the most in example 2.As evaluated by SDS-PAGE, pure
The u-FSH changed shows the single band purity (Figure 11) in gel and as evaluated by HP-SEC, pure more than 98%
Degree (Figure 12).
Claims (14)
1. a method for purification promoting sexual gland hormone, including following steps necessary:
(a) affinity chromatography；
B () anion-exchange chromatography, is followed by other suitable purification steps.
Method the most according to claim 1, wherein affinity chromatography substrate is promoting sexual gland hormone specificity affinity substrate.
Method the most according to claim 1, wherein, under the conditions of neutral buffered liquid pH or in condition of acidic pH under walk
Suddenly the eluting of the promoting sexual gland hormone after (a).
Method the most according to claim 1, wherein, anionite is selected from DEAE agarose gel, Mono Q and Q fine jade
Sepharose XL, preferably Q agarose gel.
5. the method for purification promoting sexual gland hormone, comprises the following steps:
(a) affinity chromatography；
(b) anion-exchange chromatography；
(c) hydrophobic interaction chromatograph
Wherein, step (b) and (c) can be carried out in any order.
Method the most according to claim 5, wherein, hydrophobicity base for post matter is selected from phenyl Sepharose, butyl-agarose
Gel, Octyl Sepharose, preferably phenyl Sepharose.
Method the most according to claim 5, wherein, in step (c), utilizes to the salinity of Gradient from post eluting institute
State promoting sexual gland hormone.
Method the most according to claim 7, wherein, described salt is selected from ammonium sulfate, sodium chloride, ammonium chloride and sodium sulfate,
Preferably sulfuric acid ammonium.
9. method according to the purification promoting sexual gland hormone described in arbitrary aforementioned claim from crude mixture, including following step
Rapid:
(a) cell separation and readjusting；
(b) affinity column chromatography；
(c) Ultrafiltration-Diafiltration and readjusting；
D () is virally inactivated；
(e) Anion exchange column chromatography (AEX)；
F () readjusts；
(g) hydrophobic interaction column chromatography (HIC)；
(h) Ultrafiltration-Diafiltration；
(i) nanofiltration；
(j) microfiltration
Wherein, after affinity chromatography step, hydrophobic chromatography step and anion-exchange chromatography step can be in any order
Carry out；Step (c) to (j) can be carried out in any order.
Method the most according to claim 9, wherein, filtration media selected from Tris-Cl, buffer, phosphate buffer,
Acetate buffer, citrate buffer, Succinate Buffer and combinations thereof.
11. according to the method described in claim 5 and 9, and wherein, affinity chromatography is according to the carrying out described in claim 2 and 3；Cloudy
Ion exchange chromatography is according to claim 4 to be carried out and hydrophobic interaction chromatograph is according to described in claim 6 to 8
Carrying out.
12. according to method in any one of the preceding claims wherein, wherein, described promoting sexual gland hormone be derived from cell culture or
It is derived from the crude mixture of urine.
13. according to method in any one of the preceding claims wherein, and wherein, promoting sexual gland hormone is selected from follicle stimulating hormone, rush Huang
Body generates hormone, human chorionic gonadotropin and combinations thereof.
14. according to method in any one of the preceding claims wherein, and wherein, promoting sexual gland hormone is selected from r-hFSH, u-FSH, r-
HLH, u-LH, r-hHCG and u-HCG.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IN1398MU2014 | 2014-04-18 | ||
| IN1398/MUM/2014 | 2014-04-18 | ||
| PCT/IN2015/000175 WO2015159309A1 (en) | 2014-04-18 | 2015-04-17 | Novel purification process of gonadotropin |
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| CN106170493A true CN106170493A (en) | 2016-11-30 |
Family
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|---|---|
| US (1) | US20170029482A1 (en) |
| EP (1) | EP3131924A1 (en) |
| JP (1) | JP2017514803A (en) |
| KR (1) | KR20160131113A (en) |
| CN (1) | CN106170493A (en) |
| AR (1) | AR100131A1 (en) |
| AU (1) | AU2015248461B2 (en) |
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| MX (1) | MX2016013416A (en) |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108503705A (en) * | 2018-07-10 | 2018-09-07 | 北京伟杰信生物科技有限公司 | A kind of recombination chorionic gonadotrophin（rhCG）Purification process |
| CN111303274A (en) * | 2020-03-21 | 2020-06-19 | 上海浦东明炎生物技术有限公司 | Purification method of human chorionic gonadotropin |
| CN114867741A (en) * | 2019-12-26 | 2022-08-05 | 株式会社Lg化学 | Method for purifying follicle stimulating hormone |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105968185A (en) * | 2016-07-20 | 2016-09-28 | 宁波人健药业集团股份有限公司 | Chorionic gonadotrophin purification method |
| CN114591414A (en) * | 2022-03-24 | 2022-06-07 | 江西浩然生物制药有限公司 | Preparation method of human chorionic gonadotropin |
| CN116143901A (en) * | 2022-11-28 | 2023-05-23 | 景泽生物医药(合肥)股份有限公司 | Purification method of follicle stimulating hormone |
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| EP2325194A1 (en) * | 2009-11-24 | 2011-05-25 | Glycotope GmbH | Process for the purification of glycoproteins |
| CN102464713A (en) * | 2010-12-21 | 2012-05-23 | 上海丽珠制药有限公司 | Preparation method of follicle-stimulating hormone |
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- 2015-04-17 KR KR1020167028363A patent/KR20160131113A/en active Search and Examination
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- 2015-04-17 MX MX2016013416A patent/MX2016013416A/en unknown
- 2015-04-17 EP EP15745250.9A patent/EP3131924A1/en not_active Withdrawn
- 2015-04-17 JP JP2016562233A patent/JP2017514803A/en active Pending
- 2015-04-17 BR BR112016023680A patent/BR112016023680A2/en not_active Application Discontinuation
- 2015-04-17 AR ARP150101169A patent/AR100131A1/en unknown
- 2015-04-17 WO PCT/IN2015/000175 patent/WO2015159309A1/en active Application Filing
- 2015-04-17 CN CN201580019503.6A patent/CN106170493A/en active Pending
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| CN102448979A (en) * | 2009-04-01 | 2012-05-09 | 拜奥吉耐里克斯股份公司 | Method for purifying recombinant FSH |
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Also Published As
| Publication number | Publication date |
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| SG11201608513WA (en) | 2016-11-29 |
| CA2945591A1 (en) | 2015-10-22 |
| MY183011A (en) | 2021-02-05 |
| EP3131924A1 (en) | 2017-02-22 |
| WO2015159309A1 (en) | 2015-10-22 |
| AU2015248461B2 (en) | 2017-05-04 |
| IL248293A (en) | 2017-04-30 |
| KR20160131113A (en) | 2016-11-15 |
| BR112016023680A2 (en) | 2017-08-15 |
| AU2015248461A1 (en) | 2016-11-03 |
| US20170029482A1 (en) | 2017-02-02 |
| JP2017514803A (en) | 2017-06-08 |
| MX2016013416A (en) | 2017-01-18 |
| AR100131A1 (en) | 2016-09-14 |
| EA201691834A1 (en) | 2017-02-28 |
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